It is long range goal of this project to determine the composition, function and location of the nuclear acceptor sites (nuclear binding sites) of the avian progesterone receptor (PR). Two, and possibly other, candidate chromatin acceptor proteins for PR, termed Receptor Binding Factors (RBF), have been identified. As these proteins are removed from the chromatin (along with other proteins), a loss of the specific PR binding sites for PR is observed. When bound to avian DNA, both impure and pure fractions of these proteins generate high affinity, saturable, receptor-dependent binding of the PR to DNA. One of these proteins, RBF-1, has been purified over 105x fold to apparent homogeneity. It has been partially sequenced and monoclonal antibodies (MAb) prepared against it. RBF-1 is a unique 10 kDa, hydrophobic protein with a pI ~5.5 and is species and tissue specific. The RBF-1 and the PR nuclear binding sites were both localized to the oviduct nuclear matrix using MAb in Western blot analyses and cell-free binding assays respectively. The RBF-1 activity has been distinguished from a second activity, RBF-2, which is more tightly bound to the DNA than the RBF-1 and has been under investigation for some time as a component of the nucleoacidic protein (NAP). RBF-2 does generate specific PR binding sites and is also suspected to be associated with the nuclear matrix. However, no proteins in the crude RBF-2 preparations are recognized by the anti-RBF-1 antibodies. Recent studies using Southwestern blot (and DNA gel shift) analyses indicate that RBF-1 has a high affinity for specific sequences in the matrix DNA. Preliminary evidence also suggests that these sequences may reside in or near the steroid regulated genes but not the nonregulated genes. This project proposes to further characterize RBF-1 by completing its amino acid sequence, confirm its intracellular/nuclear location using in situ techniques, determine its receptor specificity and its half-life in primary oviduct cell cultures, and assess its role in generating the high affinity class of PR binding sites. Identification of the DNA element to which the RBF-1 binds will be attempted using Southwestern and DNA gel shift assays. Its sequence will be compared to those of known steroid response elements, as well as to sequences in or around steroid regulated genes, with emphasis on the rapid steroid regulated c-myc and c-jun proto-oncogenes. The specific bases within the element to which the RBF-1 binds, and the additional bases to which the receptor (PR) itself may bind, will be examined. Lastly, the role of this element and/or its flanking domains, as steroid response element, will be examined. The isolation of the cDNA and genomic sequences of the RBF-1 protein and the analyses of the biological function of the protein will be pursued. Attempts to purify and characterize the RBF-2 will made if time permits.